Prophylactic therapy with human amniotic fluid stem cells improved survival in a rat model of lipopolysaccharide-induced neonatal sepsis through immunomodulation via aggregates with peritoneal macrophages

BACKGROUND: Despite recent advances in neonatal care, sepsis remains a leading cause of mortality in neonates. Mesenchymal stem cells derived from various tissues, such as bone marrow, umbilical cord, and adipose tissue, have beneficial effects on adult sepsis. Although human amniotic fluid stem cells (hAFSCs) have mesenchymal stem cell properties, the efficacy of hAFSCs on neonatal sepsis is yet to be elucidated. This study aimed to investigate the therapeutic potential of hAFSCs on neonatal sepsis using a rat model of lipopolysaccharide (LPS)-induced sepsis. METHODS: hAFSCs were isolated as CD117-positive cells from human amniotic fluid. Three-day-old rat pups were intraperitoneally treated with LPS to mimic neonatal sepsis. hAFSCs were administered either 3 h before or at 0, 3, or 24 h after LPS exposure. Serum inflammatory cytokine levels, gene expression profiles from spleens, and multiple organ damage were analyzed. hAFSC localization was determined in vivo. In vitro LPS stimulation tests were performed using neonatal rat peritoneal macrophages co-cultured with hAFSCs in a cell-cell contact-dependent/independent manner. Immunoregulation in the spleen was determined using a DNA microarray analysis. RESULTS: Prophylactic therapy with hAFSCs improved survival in the LPS-treated rats while the hAFSCs transplantation after LPS exposure did not elicit a therapeutic response. Therefore, hAFSC pretreatment was used for all subsequent studies. Inflammatory cytokine levels were elevated after LPS injection, which was attenuated by hAFSC pretreatment. Subsequently, inflammation-induced damages in the brain, lungs, and liver were ameliorated. hAFSCs aggregated with peritoneal macrophages and/or transiently accumulated in the liver, mesentery, and peritoneum. Paracrine factors released by hAFSCs induced M1-M2 macrophage polarization in a cell-cell contact-independent manner. Direct contact between hAFSCs and peritoneal macrophages further enhanced the polarization. Microarray analysis of the spleen showed that hAFSC pretreatment reduced the expression of genes involved in apoptosis and inflammation and subsequently suppressed toll-like receptor 4 signaling pathways. CONCLUSIONS: Prophylactic therapy with hAFSCs improved survival in a rat model of LPS-induced neonatal sepsis. These effects might be mediated by a phenotypic switch from M1 to M2 in peritoneal macrophages, triggered by hAFSCs in a cell-cell contact-dependent/independent manner and the subsequent immunomodulation of the spleen.